Cotton cultivar DP 6222 RR acala

ABSTRACT

A cotton cultivar, designated DP 6222 RR Acala, is disclosed. The invention relates to the seeds of cotton cultivar DP 6222 RR Acala, to the plants of cotton DP 6222 RR Acala and to methods for producing a cotton plant produced by crossing the cultivar DP 6222 RR Acala with itself or another cotton variety. The invention also relates to methods for producing a cotton plant containing in its genetic material one or more transgenes and to the transgenic cotton plants and plant parts produced by those methods. This invention also relates to cotton cultivars or breeding cultivars and plant parts derived from cotton variety DP 6222 RR Acala, to methods for producing other cotton cultivars, lines or plant parts derived from cotton cultivar DP 6222 RR Acala and to the cotton plants, varieties, and their parts derived from use of those methods. The invention further relates to hybrid cotton seeds and plants produced by crossing the cultivar DP 6222 RR Acala with another cotton cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a cotton (Gossypium) seed, a cottonplant, a cotton cultivar and a cotton hybrid. This invention furtherrelates to a method for producing cotton seed and plants. Allpublications cited in this application are herein incorporated byreference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single cultivar animproved combination of desirable traits from the parental germplasm. Incotton, the important traits include higher fiber (lint) yield, earliermaturity, improved fiber quality, resistance to diseases and insects,resistance to drought and heat, and improved agronomic traits.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination and the numberof hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared topopular cultivars in environments representative of the commercialtarget area(s) for three or more years. The best lines havingsuperiority over the popular cultivars are candidates to become newcommercial cultivars. Those lines still deficient in a few traits arediscarded or utilized as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, usually take from seven to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior because, for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental lines and widely grown standardcultivars. For many traits a single observation is inconclusive, andreplicated observations over time and space are required to provide agood estimate of a line's genetic worth.

The goal of a commercial cotton breeding program is to develop new,unique and superior cotton cultivars. The breeder initially selects andcrosses two or more parental lines, followed by generation advancementand selection, thus producing many new genetic combinations. The breedercan theoretically generate billions of different genetic combinationsvia this procedure. The breeder has no direct control over which geneticcombinations will arise in the limited population size which is grown.Therefore, two breeders will never develop the same line having the sametraits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The lines whichare developed are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce, with any reasonable likelihood, thesame cultivar twice by using the exact same original parents and thesame selection techniques. This unpredictability results in theexpenditure of large amounts of research moneys to develop superior newcotton cultivars.

Pureline cultivars of cotton are commonly bred by hybridization of twoor more parents followed by selection. The complexity of inheritance,the breeding objectives and the available resources influence thebreeding method. Pedigree breeding, recurrent selection breeding andbackcross breeding are breeding methods commonly used in self pollinatedcrops such as cotton. These methods refer to the manner in whichbreeding pools or populations are made in order to combine desirabletraits from two or more cultivars or various broad-based sources. Theprocedures commonly used for selection of desirable individuals orpopulations of individuals are called mass selection, plant-to-rowselection and single seed descent or modified single seed descent. One,or a combination of these selection methods, can be used in thedevelopment of a cultivar from a breeding population.

Pedigree breeding is primarily used to combine favorable genes into atotally new cultivar that is different in many traits than either parentused in the original cross. It is commonly used for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁ (filial generation 1).An F₂ population is produced by selfing F₁ plants. Selection ofdesirable individual plants may begin as early as the F₂ generationwherein maximum gene segregation occurs. Individual plant selection canoccur for one or more generations. Successively, seed from each selectedplant can be planted in individual, identified rows or hills, known asprogeny rows or progeny hills, to evaluate the line and to increase theseed quantity, or, to further select individual plants. Once a progenyrow or progeny hill is selected as having desirable traits it becomeswhat is known as a breeding line that is specifically identifiable fromother breeding lines that were derived from the same originalpopulation. At an advanced generation (i.e., F₅ or higher) seed ofindividual lines are evaluated in replicated testing. At an advancedstage the best lines or a mixture of phenotypically similar lines fromthe same original cross are tested for potential release as newcultivars.

The single seed descent procedure in the strict sense refers to plantinga segregating population, harvesting one seed from every plant, andcombining these seeds into a bulk which is planted the next generation.When the population has been advanced to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. Primary advantages of the seed descentprocedures are to delay selection until a high level of homozygosity(e.g., lack of gene segregation) is achieved in individual plants, andto move through these early generations quickly, usually through usingwinter nurseries.

The modified single seed descent procedures involve harvesting multipleseed (i.e., a single lock or a simple boll) from each plant in apopulation and combining them to form a bulk. Part of the bulk is usedto plant the next generation and part is put in reserve. This procedurehas been used to save labor at harvest and to maintain adequate seedquantities of the population.

Selection for desirable traits can occur at any segregating generation(F₂ and above). Selection pressure is exerted on a population by growingthe population in an environment where the desired trait is maximallyexpressed and the individuals or lines possessing the trait can beidentified. For instance, selection can occur for disease resistancewhen the plants or lines are grown in natural or artificially-induceddisease environments, and the breeder selects only those individualshaving little or no disease and are thus assumed to be resistant.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (Molecular Linkage Map ofSoybean (Glycine max L. Merr.) p 6.131-6.138 in S. J. O'Brien (ed)Genetic Maps: Locus Maps of Complex Genomes, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1993)) developed amolecular genetic linkage map that consisted of 25 linkage groups withabout 365 RFLP, 11 RAPD and three classical markers and four isozymeloci. See also, Shoemaker, R. C., RFLP Map of Soybean, p 299-309, inPhillips, R. L. and Vasil, I. K. (eds.) DNA-Based Markers in Plants,Kluwer Academic Press, Dordrecht, the Netherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. (Diwan, N. and Cregan, P.B., Theor. Appl. Genet. 95:22-225, 1997.) SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. For example, molecularmarkers are used in soybean breeding for selection of the trait ofresistance to soybean cyst nematode, see U.S. Pat. No. 6,162,967. Themarkers can also be used to select toward the genome of the recurrentparent and against the markers of the donor parent. Using this procedurecan attempt to minimize the amount of genome from the donor parent thatremains in the selected plants. It can also be used to reduce the numberof crosses back to the recurrent parent needed in a backcrossingprogram. The use of molecular markers in the selection process is oftencalled Genetic Marker Enhanced Selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses as discussed more fully hereinafter.

Mutation breeding is another method of introducing new traits intocotton varieties. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, radiation (such as X-rays, Gamma rays, neutrons,Beta radiation, or ultraviolet radiation), chemical mutagens (such asbase analogues like 5-bromo-uracil), antibiotics, alkylating agents(such as sulfur mustards, nitrogen mustards, epoxides, ethyleneamines,sulfates, sulfonates, sulfones, or lactones), azide, hydroxylamine,nitrous acid or acridines. Once a desired trait is observed throughmutagenesis the trait may then be incorporated into existing germplasmby traditional breeding techniques. Details of mutation breeding can befound in “Principles of Cultivar Development” by Fehr, MacmillanPublishing Company, 1993.

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan et al., Theor. Appl. Genet., 77:889-892, 1989.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep, et al. 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, and the grower, processor and consumer; forspecial advertising and marketing and commercial production practices,and new product utilization. The testing preceding the release of a newcultivar should take into consideration research and development costsas well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Cotton, Gossypium hirsutum, is an important and valuable field crop.Thus, a continuing goal of cotton plant breeders is to develop stable,high yielding cotton cultivars that are agronomically sound. The reasonsfor this goal are obviously to maximize the amount and quality of thefiber produced on the land used and to supply fiber, oil and food foranimals and humans. To accomplish this goal, the cotton breeder mustselect and develop plants that have the traits that result in superiorcultivars.

The development of new cotton cultivars requires the evaluation andselection of parents and the crossing of these parents. The lack ofpredictable success of a given cross requires that a breeder, in anygiven year, make several crosses with the same or different breedingobjectives.

The cotton flower is monoecious in that the male and female structuresare in the same flower. The crossed or hybrid seed is produced by manualcrosses between selected parents. Floral buds of the parent that is tobe the female are emasculated prior to the opening of the flower bymanual removal of the male anthers. At flowering, the pollen fromflowers of the parent plants designated as male, are manually placed onthe stigma of the previous emasculated flower. Seed developed from thecross is known as first generation (F₁) hybrid seed. Planting of thisseed produces F₁ hybrid plants of which half their genetic component isfrom the female parent and half from the male parent. Segregation ofgenes begins at meiosis thus producing second generation (F₂) seed.Assuming multiple genetic differences between the original parents, eachF₂ seed has a unique combination of genes.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods which aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

The present invention relates to a cotton seed, a cotton plant, a cottonvariety and a method for producing a cotton plant.

The present invention further relates to a method of producing cottonseeds and plants by crossing a plant of the instant invention withanother cotton plant.

This invention further relates to the seeds of cotton variety DP 6222 RRAcala, to the plants of cotton variety DP 6222 RR Acala and to methodsfor producing a cotton plant produced by crossing the cotton DP 6222 RRAcala with itself or another cotton cultivar. Thus, any such methodsusing the cotton variety DP 6222 RR Acala are part of this invention,including selfing, backcrosses, hybrid production, crosses topopulations, and the like.

In another aspect, the present invention provides for single traitconverted plants of DP 6222 RR Acala. The single transferred trait maypreferably be a dominant or recessive allele. Preferably, the singletransferred trait will confer such traits as herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, male sterility, enhanced fiber quality, and industrialusage. The single trait may be a naturally occurring cotton gene or atransgene introduced through genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of cotton plant DP 6222 RR Acala. The tissueculture will preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing cottonplant, and of regenerating plants having substantially the same genotypeas the foregoing cotton plant. Preferably, the regenerable cells in suchtissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, orstems. Still further, the present invention provides cotton plantsregenerated from the tissue cultures of the invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. Allele is any of one or more alternative forms of a gene, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Disease Resistance. As used herein, the term “disease resistance” isdefined as the ability of plants to restrict the activities of aspecified pest, such as an insect, fungus, virus, or bacterial.

Disease Tolerance. As used herein, the term “disease tolerance” isdefined as the ability of plants to endure a specified pest (such as aninsect, fungus, virus or bacteria) or an adverse environmental conditionand still perform and produce in spite of this disorder.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted trait.

Fallout (Fo). As used herein, the term “fallout” refers to the rating ofhow much cotton has fallen on the ground at harvest.

Fiber Elongation (E1). As used herein, the term “fiber elongation” isdefined as the measure of elasticity of a bundle of fibers as measuredby HVI.

Fiber Length (Len). As used herein, the term “fiber length” is definedas 2.5% span length in inches of fiber as measured by High VolumeInstrumentation (HVI).

Fiber Strength (T1). As used herein, the term “fiber strength” isdefined as the force required to break a bundle of fibers as measured ingrams per millitex on the HVI.

Fruiting Nodes. As used herein, the term “fruiting nodes” is defined asthe number of nodes on the main stem from which arise branches whichbear fruit or bolls.

Gin Turnout. As used herein, the term “gin turnout” is defined as afraction of lint in a machine harvested sample of seed cotton (lint,seed, and trash).

Lint/boll. As used herein, the term “lint/boll” is the weight of lintper boll.

Lint Index. As used herein, the term “lint index” refers to the weightof lint per seed in milligrams.

Lint Percent. As used herein, the term “lint percent” is defined as thelint (fiber) fraction of seed cotton (lint and seed).

Lint Yield (Lint %). As used herein, the term “lint yield” is defined asthe measure of the quantity of fiber produced on a given unit of land.Presented below in kilograms of lint per hectare.

Maturity Rating (Mat). As used herein, the term “maturity rating” isdefined as a visual rating near harvest on the amount of opened bolls onthe plant.

Micronaire (Mic). As used herein, the term “micronaire” is defined as ameasure of the fineness of the fiber. Within a cotton cultivar,micronaire is also a measure of maturity. Micronaire differences aregoverned by changes in perimeter or in cell wall thickness, or bychanges in both. Within a variety, cotton perimeter is fairly constantand maturity will cause a change in micronaire. Consequently, micronairehas a high correlation with maturity within a variety of cotton.Maturity is the degree of development of cell wall thickness. Micronairemay not have a good correlation with maturity between varieties ofcotton having different fiber perimeter. Micronaire values range fromabout 2.0 to 6.0:

Below 2.9 Very Possible small perimeter but mature (good fiber), fine orlarge perimeter but immature (bad fiber). 2.9 to 3.7 Fine Variousdegrees of maturity and/or perimeter. 3.8 to 4.6 Average Average degreeof maturity and/or perimeter. 4.7 to 5.5 Coarse Usually fully developed(mature), but larger perimeter. 5.6 + Very Fully developed,large-perimeter fiber. coarse

Plant Height (Hgt). As used herein, the term “plant height” is definedas the average height in inches or centimeters of a group of plants.

Seed/boll. As used herein, the term “seed/boll” refers to the number ofseeds per boll.

Seedcotton/boll. As used herein, the term “seedcotton/boll” refers tothe weight of seedcotton per boll.

Seedweight (Sdwt). As used herein, the term “seedweight” is the weightof 100 seeds in grams.

Single trait Converted (Conversion). Single trait converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing or via genetic engineering wherein essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the single trait transferred intothe variety via the backcrossing technique or via genetic engineering.

Stringout Rating (So). As used herein, the term “stringout rating” isdefined as a visual rating prior to harvest of the relative looseness ofthe seed cotton held in the boll structure on the plant.

Uniformity Ratio (UR). As used herein, the term “uniformity ratio” isdefined as a measure of the relative length uniformity of a bundle offibers as measured by HVI.

Vegetative Nodes. As used herein, the term “vegetative nodes” is definedas the number of nodes from the cotyledonary node to the first fruitingbranch on the main stem of the plant.

VRDP. As used herein, the term “VRDP” is defined as the alleledesignation for the single dominant allele of the present inventionwhich confers virus resistance. VRDP designates “Virus ResistanceDeltapine”.

DETAILED DESCRIPTION OF THE INVENTION

DP 6222 RR Acala is a mid-to-full maturity Acala picker-type uplandcotton variety. DP 6222 RR Acala has the gene insertion line 1445 of aconstruct developed by the Monsanto Company which causes these plants tobe tolerant to the herbicide ROUNDUP (glyphosate).

The cultivar has shown uniformity and stability, as described in thefollowing variety description information. It has been self-pollinated asufficient number of generations with careful attention to uniformity ofplant type. The cultivar has been increased with continued observationfor uniformity.

Cotton cultivar DP 6222 RR Acala has the following morphologic and othercharacteristics.

TABLE I VARIETY DESCRIPTION INFORMATION Species: Gossypium hirsutum L.General: Plant Habit: Intermediate Foliage: Intermediate Stem Lodging:Erect Fruiting Branch: Short Growth: Intermediate Leaf Color: Dark greenBoll Shape: Length equal to width Boll Breadth: Broadest at middleMaturity: Date of 50% open bolls: 171.5 Plant: Cm to 1st Fruiting Branch(from cotyledonary  15.3 node): No. of Nodes to 1st Fruiting Branch(excluding  5.8 cotyledonary node): Mature Plant Height (fromcotyledonary node to 119.9 cm terminal): Leaf (Upper most, fullyexpanded leaf): Type: Normal Pubescence: Medium Nectaries: Present StemPubescence: Intermediate Glands: Leaf: Normal Stem: Normal Calyx lobe:Normal Flower: Petals: Cream Pollen: Cream Petal spot: Absent Seed: SeedIndex (g/100 seed, fuzzy basis):  13.3 Lint Index: (g lint/100 seeds): 9.0 Boll: Lint percent, picked:  40.3 Number of seeds per boll:  30.2Grams seed cotton per boll:  6.3 Boll Type: Open Fiber Properties:Length (inches, 2.5% SL):  1.185 Uniformity (%):  83.9 Strength, T1(g/tex):  36.5 Elongation, E1 (%):  11.4 Micronaire:  4.4

In Table 2 below, DP 6222 RR Acala is compared with commercial varietyAcala SIERRA RR for several characteristics. DP 6222 RR Acala and AcalaSIERRA RR are similar, however there are numerous differences. As can beseen in Table 2, there are significant differences from the comparisonvariety at the 5% level of probability or less.

TABLE 2 DP 6222 RR Acala SIERRA Trait Acala RR Lint turnout 0.403 0.417No. Of Nodes to First Fruiting Branch 5.8 6.7 Seed Index 13.3 12.1 LintIndex 9.0 8.6

This invention is also directed to methods for producing a cotton plantby crossing a first parent cotton plant with a second parent cottonplant, wherein the first or second cotton plant is the cotton plant fromthe cultivar DP 6222 RR Acala. Further, both the first and second parentcotton plants may be the cultivar DP 6222 RR Acala (e.g.,self-pollination). Therefore, any methods using the cultivar DP 6222 RRAcala are part of this invention: selfing, backcrosses, hybrid breeding,and crosses to populations. Any plants produced using cultivar DP 6222RR Acala as a parent are within the scope of this invention. As usedherein, the term “plant” includes plant cells, plant protoplasts, plantcells of tissue culture from which cotton plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants, such as pollen, flowers, embryos, ovules, seeds, pods,leaves, stems, roots, anthers and the like. Thus, another aspect of thisinvention is to provide for cells which upon growth and differentiationproduce a cultivar having essentially all of the physiological andmorphological characteristics of DP 6222 RR Acala.

The present invention contemplates a cotton plant regenerated from atissue culture of a variety (e.g., DP 6222 RR Acala) or hybrid plant ofthe present invention. As is well known in the art, tissue culture ofcotton can be used for the in vitro regeneration of a cotton plant.Tissue culture of various tissues of cotton and regeneration of plantstherefrom is well known and widely published.

Further Embodiments of the Invention

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed variety or cultivar.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed cotton plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the cotton plant(s).

Expression Vectors for Cotton Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII), which, when under the control ofplant regulatory signals confers resistance to kanamycin. Fraley et al.,Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Another commonly usedselectable marker gene is the hygromycin phosphotransferase gene whichconfers resistance to the antibiotic hygromycin. Vanden Elzen et al.,Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990) Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvyl-shikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes publication2908, Imagene Green, p. 1-4 (1993) and Naleway et al., J. Cell Biol.115:151a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Cotton Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in cotton. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in cotton. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in cotton or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in cotton.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application WO 96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in cotton.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in cotton. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter—such as that from the phaseolin gene (Murai et al., Science23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci.U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promotersuch as that from cab or rubisco (Simpson et al., EMBO J.4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); ananther-specific promoter such as that from LAT52 (Twell et al., Mol.Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such asthat from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993))or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al., PlantMol. Biol. 9:3-17 (1987); Lerner et al., Plant Physiol. 91:124-129(1989); Fontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal., Cell 39:499-509 (1984); Steifel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a cotton plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A gene conferring resistance to a pest, such as nematodes. See e.g.,PCT Application WO 96/30517; PCT Application WO 93/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

D. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See PCT application WO 95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

S. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to an Herbicide:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT, bar, genes), and pyridinoxy or phenoxy proprionicacids and cyclohexones (ACCase inhibitor-encoding genes). See, forexample, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses thenucleotide sequence of a form of EPSP which can confer glyphosateresistance. A DNA molecule encoding a mutant aroA gene can be obtainedunder ATCC accession number 39256, and the nucleotide sequence of themutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Europeanpatent application No. 0 333 033 to Kumada et al., and U.S. Pat. No.4,975,374 to Goodman et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a PAT gene is provided inEuropean application No. 0 242 246 to Leemans et al. DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for PAT activity. Exemplary ofgenes conferring resistance to phenoxy proprionic acids andcyclohexones, such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2and Acc1-S3 genes described by Marshall et al., Theor. Appl. Genet.83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibila et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B. Decreased phytate content—1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished, by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus licheniformis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

4. Genes that Control Male Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622, 1992).

Methods for Cotton Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

B. Direct Gene Transfer—Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation wherein DNA is carried on the surface of microprojectilesmeasuring 1 to 4 μm. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987),Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206(1990), Klein et al., Biotechnology 10:268 (1992). See also U.S. Pat.No. 5,015,580 (Christou, et al.), issued May 14, 1991; U.S. Pat. No.5,322,783 (Tomes, et al.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues has also beendescribed. Donn et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994).

Following transformation of cotton target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed, with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular cotton cultivar using theforegoing transformation techniques could be moved into another cultivarusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Single-Gene Conversion

When the term “cotton plant” is used in the context of the presentinvention, this also includes any single gene conversions of thatvariety. The term “single gene converted plant” as used herein refers tothose cotton plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the variety. The term“backcrossing” as used herein refers to the repeated crossing of ahybrid progeny back to the recurrent parent, i.e., backcrossing 1, 2, 3,4, 5, 6, 7, 8, 9 or more times to the recurrent parent. The parentalcotton plant which contributes the gene for the desired characteristicis termed the “nonrecurrent” or “donor parent”. This terminology refersto the fact that the nonrecurrent parent is used one time in thebackcross protocol and therefore does not recur. The parental cottonplant to which the gene or genes from the nonrecurrent parent aretransferred is known as the recurrent parent as it is used for severalrounds in the backcrossing protocol (Poehlman & Sleper, 1994; Fehr,1987). In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single gene of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a cotton plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred gene from thenonrecurrent parent, as determined at the 5% significance level whengrown in the same environmental conditions.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross. One ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic, examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185, 5,973,234 and 5,977,445, the disclosures of which arespecifically hereby incorporated by reference.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of cotton andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Komatsuda, T. et al., Crop Sci.31:333-337 (1991); Stephens, P. A., et al., Theor. Appl. Genet.82:633-635 (1991); Komatsuda, T. et al., Plant Cell, Tissue and OrganCulture, 28:103-113 (1992); Dhir, S. et al. Plant Cell Reports11:285-289 (1992); Pandey, P. et al., Japan J. Breed. 42:1-5 (1992); andShetty, K., et al., Plant Science 81:245-251 (1992); as well as U.S.Pat. No. 5,024,944 issued Jun. 18, 1991 to Collins et al., and U.S. Pat.No. 5,008,200 issued Apr. 16, 1991 to Ranch et al. Thus, another aspectof this invention is to provide cells which upon growth anddifferentiation produce cotton plants having the physiological andmorphological characteristics of cotton variety DP 6222 RR Acala.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234 and 5,977,445,described certain techniques.

This invention also is directed to methods for producing a cotton plantby crossing a first parent cotton plant with a second parent cottonplant wherein the first or second parent cotton plant is a cotton plantof the variety DP 6222 RR Acala. Further, both first and second parentcotton plants can come from the cotton variety DP 6222 RR Acala. Thus,any such methods using the cotton variety DP 6222 RR Acala are part ofthis invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cotton variety DP6222 RR Acala as a parent are within the scope of this invention,including those developed from varieties derived from cotton variety DP6222 RR Acala. Advantageously, the cotton variety could be used incrosses with other, different, cotton plants to produce first generation(F₁) cotton hybrid seeds and plants with superior characteristics. Thevariety of the invention can also be used for transformation whereexogenous genes are introduced and expressed by the variety of theinvention. Genetic variants created either through traditional breedingmethods using variety DP 6222 RR Acala or through transformation of DP6222 RR Acala by any of a number of protocols known to those of skill inthe art are intended to be within the scope of this invention.

The following describes breeding methods that may be used with cultivarDP 6222 RR Acala in the development of further cotton plants. One suchembodiment is a method for developing an DP 6222 RR Acala progeny cottonplant in a cotton plant breeding program comprising: obtaining thecotton plant, or a part thereof, of cultivar DP 6222 RR Acala utilizingsaid plant or plant part as a source of breeding material and selectingan DP 6222 RR Acala progeny plant with molecular markers in common withDP 6222 RR Acala and/or with morphological and/or physiologicalcharacteristics selected from the characteristics listed in Tables 1 or2. Breeding steps that may be used in the cotton plant breeding programinclude pedigree breeding, back crossing, mutation breeding, andrecurrent selection. In conjunction with these steps, techniques such asRFLP-enhanced selection, genetic marker enhanced selection (for exampleSSR markers) and the making of double haploids may be utilized.

Another method involves producing a population of cultivar DP 6222 RRAcala progeny cotton plants, comprising crossing cultivar DP 6222 RRAcala with another cotton plant, thereby producing a population ofcotton plants, which, on average, derive 50% of their alleles fromcultivar DP 6222 RR Acala. A plant of this population may be selectedand repeatedly selfed or sibbed with a cotton cultivar resulting fromthese successive filial generations. One embodiment of this invention isthe cotton cultivar produced by this method and that has obtained atleast 50% of its alleles from cultivar DP 6222 RR Acala.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the invention includes cottoncultivar DP 6222 RR Acala progeny cotton plants comprising a combinationof at least two DP 6222 RR Acala traits selected from the groupconsisting of those listed in Tables 1 and 2 or the DP 6222 RR Acalacombination of traits listed in the Summary of the Invention, so thatsaid progeny cotton plant is not significantly different for said traitsthan cotton cultivar DP 6222 RR Acala as determined at the 5%significance level when grown in the same environment. Using techniquesdescribed herein, molecular markers may be used to identify said progenyplant as a DP 6222 RR Acala progeny plant. Mean trait values may be usedto determine whether trait differences are significant, and preferablythe traits are measured on plants grown under the same environmentalconditions. Once such a variety is developed its value is substantialsince it is important to advance the germplasm base as a whole in orderto maintain or improve traits such as yield, disease resistance, pestresistance, and plant performance in extreme environmental conditions.

Progeny of cultivar DP 6222 RR Acala may also be characterized throughtheir filial relationship with cotton cultivar DP 6222 RR Acala, as forexample, being within a certain number of breeding crosses of cottoncultivar DP 6222 RR Acala. A breeding cross is a cross made to introducenew genetics into the progeny, and is distinguished from a cross, suchas a self or a sib cross, made to select among existing genetic alleles.The lower the number of breeding crosses in the pedigree, the closer therelationship between cotton cultivar DP 6222 RR Acala and its progeny.For example, progeny produced by the methods described herein may bewithin 1, 2, 3, 4 or 5 breeding crosses of cotton cultivar DP 6222 RRAcala.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which cotton plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, pods, leaves,roots, root tips, anthers, and the like.

Tables

As shown in Table 3 below, DP 6222 RR Acala is compared to commercialcotton variety Acala SIERRA RR. Column one shows the repetition numberfor the site, column 2 shows the site abbreviation, column 3 shows thenumber of centimeters to the first fruiting branch, column 4 shows thenumber of nodes to the first fruiting branch, column 5 shows the plantheight in centimeters, column 6 shows the maturity, column 7 shows theseed index, column 8 shows the lint index, column 9 shows the number ofseeds per boll, and column 10 shows the grams of seed cotton per boll.

TABLE 3 Comparison of Select Plant Characteristics Between DP 6222 RRAcala and Acala Sierra RR for Year 2004 Cm 1^(st) No. Nodes PlantMaturity Fruiting to 1^(st) F. Height (Days 50% Seed Lint No. Seed GramsSeed Branch Branch Cm Open) Index Index per Boll Cotton/Boll DP DP DP DPDP DP DP DP 6222 Acala 6222 Acala 6222 Acala 6222 Acala 6222 Acala 6222Acala 6222 Acala 6222 Acala RR Sierra RR Sierra RR Sierra RR Sierra RRSierra RR Sierra RR Sierra RR Sierra Rep Site Acala RR Acala RR Acala RRAcala RR Acala RR Acala RR Acala RR Acala RR 1 CG 18.0 18.0 5.6 5.6143.8 111.8 13.8 12.4 8.9 8.5 29.6 34.9 5.8 6.2 2 CG 15.0 13.8 5.6 6.4139.4 136.9 13.4 11.9 9.0 8.5 34.0 33.8 6.5 5.9 3 CG 12.8 14.6 6.0 7.2121.9 135.6 13.7 12.2 8.9 8.7 35.3 32.1 6.8 5.7 1 V 6.1 7.5 100.6 116.3171 169 13.3 10.9 9.1 8.4 2 V 5.7 6.7 94.0 101.9 172 168 12.8 11.3 8.88.0 3 V 13.5 12.9 8.7 8.9 4 V 12.9 11.3 8.6 8.3 5 V 13.0 11.7 9.3 8.0 6V 13.5 11.9 9.2 8.2 1 Y 13.1 12.6 9.3 9.3 29.8 25.4 6.7 5.6 2 Y 13.012.9 9.1 9.2 24.9 25.2 5.5 5.6 3 Y 13.6 12.8 9.3 9.5 27.9 24.8 6.4 5.6Ave. 15.3 15.5 5.8 6.7 119.9 120.5 171.5 168.5 13.3 12.1 9.0 8.6 30.229.4 6.3 5.8 CG = Casa Grande, AZ; V = Visalia, CA; Y = Yuma, AZ

DEPOSIT INFORMATION

A deposit of the D&PL Technology Holding Company, LLC proprietary cottoncultivar DP 6222 RR Acala disclosed above and recited in the appendedclaims has been made with the American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110. The date of deposit wasSep. 2, 2005. The deposit of 2,500 seeds was taken from the same depositmaintained by the D&PL Technology Holding Company, LLC since prior tothe filing date of this application. All restrictions upon the deposithave been removed, and the deposit is intended to meet all of therequirements of 37 C.F.R. §1.801-1.809. The ATCC accession number isPTA-6966. The deposit will be maintained in the depository for a periodof 30 years, or 5 years after the last request, or for the effectivelife of the patent, whichever is longer, and will be replaced asnecessary during that period.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. A seed of cotton cultivar designated DP 6222 RR Acala, wherein arepresentative sample of seed of said cultivar was deposited under ATCCAccession No. PTA-6966.
 2. A cotton plant, or a regenerable partthereof, produced by growing the seed of claim
 1. 3. A tissue culture ofcells produced from the plant of claim 2, wherein said cells of thetissue culture are produced from a plant part selected from the groupconsisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematiccell, root, root tip, pistil, anther, flower, stem and boll.
 4. Aprotoplast produced from the plant of claim
 2. 5. A protoplast producedfrom the tissue culture of claim
 3. 6. A cotton plant regenerated fromthe tissue culture of claim 3, wherein the plant has all of themorphological and physiological characteristics of cultivar DP 6222 RRAcala.
 7. A method for producing an F₁ hybrid cotton seed, wherein themethod comprises crossing the plant of claim 2 with a different cottonplant and harvesting the resultant F₁ hybrid cotton seed.
 8. A method ofproducing an herbicide resistant cotton plant wherein the methodcomprises transforming the cotton plant of claim 2 with a transgenewherein the transgene confers resistance to an herbicide selected fromthe group consisting of imidazolinone, sulfonylurea, glyphosate,glufosinate, L-phosphinothricin, triazine and benzonitrile.
 9. Anherbicide resistant cotton plant produced by the method of claim
 8. 10.A method of producing an insect resistant cotton plant wherein themethod comprises transforming the cotton plant of claim 2 with atransgene that confers insect resistance.
 11. An insect resistant cottonplant produced by the method of claim
 10. 12. The cotton plant of claim11, wherein the transgene encodes a Bacillus thuringiensis endotoxin.13. A method of producing a disease resistant cotton plant wherein themethod comprises transforming the cotton plant of claim 2 with atransgene that confers disease resistance.
 14. A disease resistantcotton plant produced by the method of claim
 13. 15. A method ofproducing a cotton plant with modified fatty acid metabolism or modifiedcarbohydrate metabolism wherein the method comprises transforming thecotton plant of claim 2 with a transgene encoding a protein selectedfrom the group consisting of phytase, fructosyltransferase,levansucrase, α-amylase, invertase and starch branching enzyme orencoding an antisense of stearyl-ACP desaturase.
 16. A cotton planthaving modified fatty acid metabolism or modified carbohydratemetabolism produced by the method of claim
 15. 17. A method ofintroducing a desired trait into cotton cultivar DP 6222 RR Acalawherein the method comprises: (a) crossing a DP 6222 RR Acala plant,wherein a representative sample of seed of said cultivar was depositedunder ATCC Accession No. PTA-6966, with a plant of another cottoncultivar that comprises a desired trait to produce progeny plantswherein the desired trait is selected from the group consisting of malesterility, herbicide resistance, insect resistance, modified fatty acidmetabolism, modified carbohydrate metabolism and resistance to bacterialdisease, fungal disease or viral disease; (b) selecting one or moreprogeny plants that have the desired trait to produce selected progenyplants; (c) crossing the selected progeny plants with the DP 6222 RRAcala plants to produce backcross progeny plants; (d) selecting forbackcross progeny plants that have the desired trait and all of thephysiological and morphological characteristics of cotton cultivar DP6222 RR Acala listed in Table 1 to produce selected backcross progenyplants; and (e) (e) repeating steps (c) and (d) three or more times insuccession to produce selected fourth or higher backcross progeny plantsthat comprise the desired trait and all of the physiological andmorphological characteristics of cotton cultivar DP 6222 RR Acala listedin Table
 1. 18. A cotton plant produced by the method of claim 17,wherein the plant has the desired trait and all of the physiological andmorphological characteristics of cotton cultivar DP 6222 RR Acala listedin Table
 1. 19. The cotton plant of claim 18, wherein the desired traitis herbicide resistance and the resistance is conferred to an herbicideselected from the group consisting of imidazolinone, sulfonylurea,glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.20. The cotton plant of claim 18, wherein the desired trait is insectresistance and the insect resistance is conferred by a transgeneencoding a Bacillus thuringiensis endotoxin.
 21. The cotton plant ofclaim 18, wherein the desired trait is modified fatty acid metabolism ormodified carbohydrate metabolism and said desired trait is conferred bya nucleic acid encoding a protein selected from the group consisting ofphytase, fructosyltransferase, levansucrase, α-amylase, invertase andstarch branching enzyme or encoding an antisense of stearyl-ACPdesaturase.
 22. A method of producing a male sterile cotton plantwherein the method comprises transforming the cotton plant of claim 2with a nucleic acid molecule.
 23. A male sterile cotton plant producedby the method of claim
 22. 24. A hybrid cotton seed produced by themethod of claim
 7. 25. A hybrid cotton plant produced by growing saidhybrid seed of claim 24.